Abstract

OBJECTIVES

The objectives of this study were 1) to improve the attachment of reimplanted endothelial cells (EC) using a fibrin glue, and 2) to assess the impact of endothelial reseeding on restenosis eight weeks after balloon angioplasty.

BACKGROUND

A possible mechanism contributing to restenosis after balloon angioplasty is the loss of the EC lining. Previous attempts to reseed EC had little effect due to rapid loss of the seeded cells.

METHODS

Twelve atherosclerotic rabbits were subjected to angioplasty of iliac arteries and reseeding procedure. One iliac artery was subjected to EC/glue reconstruction and a contralateral site to EC seeding without glue. The animals were sacrificed after 4 h. In another series 12 rabbits were treated in the same fashion and were restudied at eight weeks. Additionally, in 10 animals one iliac was subjected to glue treatment, and another served as control.

RESULTS

Histological examination demonstrated the ability of this method to reattach the EC/glue matrix circumferentially to 68.0 ± 6.7% of the arterial wall in comparison with 13.5 ± 3.9% reattachment after EC seeding. Morphometry at eight weeks showed that the lumen area was significantly greater in the EC/glue group (1.23 ± 0.35 mm2) than in the EC seeding alone (0.65 ± 0.02 mm2) and 0.72 ± 0.41 mm2 in the glue group. This was principally accounted for by the statistically significant differences in the intimal area (0.76 ± 0.18 mm2 vs. 1.25 ± 0.26 mm2 and 1.01 ± 0.53 mm2, respectively).

CONCLUSIONS

The attachment of EC after angioplasty can be greatly improved with fibrin glue matrix. The near 70% endothelial coverage achieved by this method resulted in a significant reduction of restenosis in atherosclerotic rabbit.

Despite the fact that percutaneous transluminal coronary angioplasty (PTCA) has revolutionized the treatment of coronary artery disease, restenosis of coronary vessels occurs in 30% to 50% of patients (1) within the first six months and severely limits the benefits of this well-established procedure.

It has been hypothesized that an important mechanism contributing to restenosis is the denudation of the endothelial cell (EC) lining of the arterial wall at the time of balloon angioplasty (2). Previous attempts to reseed ECs using a variety of delivery devices to the vascular wall have been hampered by rapid loss of the seeded cells (3–8).

Previous studies have demonstrated that complete circumferential reseeding of the denuded arterial wall with viable ECs is difficult with current technology, primarily because of the difficulty maintaining cell adherence when blood flow is restored (9–16).

Recently, modified autologous cryoprecipitate has become an important reagent in the ongoing attempts to line the luminal surface of vascular prosthesis with autologous ECs (13,17,18). We hypothesized that this fibrin glue may improve the efficacy of endoluminal EC seeding and reduce restenosis after PTCA.

The objective of this study was: 1) to improve the extent of initial EC reattachment to the native arteries using a fibrin glue, and 2) to assess the impact of improved endothelial reseeding on restenosis at eight weeks after balloon angioplasty.

Methods

Preparation of allogenic ECs

A 20-cm section of donor rabbit thoracic aorta was opened and rinsed with serum-free medium and then placed intimal side down on a sterile plate containing 0.2% collagenase type I (Worthington Biochemical, Freehold, New Jersey) and incubated at 37°C for 30 min. Cells were gathered by gentle abrasion with a cell scraper (CoStar, Pleasanton, California) from the edges to the center of the specimen. The cells were rinsed with 10 ml of serum-free medium into a polypropylene test tube and centrifuged for 10 min. The supernatant was removed and the cells resuspended with 10 to 20 ml of fresh complete growth medium consisting of Dulbecco’s modified Eagle’s medium (DMEM: Irvine Scientific, Santa Ana, California), supplemented with 15% heat-inactivated fetal bovine serum (Hyclone Labs, Logan, Utah), 100 U/ml penicillin, 100 U/ml streptomycin, 1 mM sodium pyruvate, 0.25 ug/ml fungizone (Irvine Scientific), 10 ug/ml EC growth supplement (Collaborative Research, Bedford, Massachusetts), 7.5 U/ml heparin (Elkins & Sinn, Cherry Hill, New Jersey), 25 mM N-2-hydroxyethyliperazine-N′2-ethanesulfonic acid buffer and 3.7 g/l sodium bicarbonate adjusted to pH 7.25. The cells were then seeded on collagen-coated T25 flasks and grown for two to four days in complete medium at 37°C in a 5% CO2 atmosphere. The cells were rinsed with phosphate-buffered saline and supplied with fresh medium on the second day. Cultures prepared by this method contained at least 95% EC (19).

Preparation of autologous ECs

Autologous ECs were harvested from one of the rabbit’s jugular veins. After removal, the vessels were immersed in Hanks solution supplemented with penicillin/streptomycin and fungizon and were immediately transported to the cell culture laboratory where they were opened longitudinally. The vein was laid down onto a Petri dish, irrigated with a few drops of 0.2% solution of collagenase (type 1A, Sigma) and incubated at 37°C for 15 min. Then the vein was washed with medium containing serum and centrifuged at 600 rpm for 5 min. The pellet was resuspended in DMEM, supplemented with serum and plated into a T25 culture flask.

Fluorescence labeling of ECs

A single cell suspension (about 0.10 ml) was incubated with 0.1 ml of a fluorescent cell marker—PKH-26 (Sigma, St. Louis, Missouri) at a concentration of 4 × 10−6 M for 5 min at 25°C. After this staining period, an equal volume of 5% albumin was added, and the mixture was centrifuged to separate the cells from the staining solution. For final washing the resuspended cells were transferred without vortexing to a new tube containing 5% albumin and centrifuged. Final cell concentration was evaluated twice in a Hemocytometer and in a spectrofluorimeter. This cellular marker (PKH-26) is retained in the original cell or its progeny for many cell cycles; in rabbit erythrocytes in vitro, this marker has shown persistent fluorescence for 60 days.

Based on concomitant measurement of fluorescence intensities and direct cell counts, a calibration curve was established by plating stained cells into wells with a surface area of 1.88cm2. After 6 h, medium and nonattached cells were removed, and the fluorescent stain, PKH-26, was completely extracted with 1.0 ml of 100% ethanol for 30 min. Intensity of fluorescence in the extracted PKH-26 solution was measured at excitation and emission wavelengths of 488 and 551 nm, respectively. Enumeration of the relative percentage of cells attached to the vessel wall was performed by comparison of intensities of this extracted stain from the cells seeded on tissue culture plates with the stain extracted from the vessel wall in similar fashion after EC/glue application and exposition to blood flow.

Preparation of fibrinogen

Standard cryoprecipitate techniques were used to prepare autologous fibrinogen from rabbit plasma collected one week before EC seeding (20,21). Whole blood was collected in 15 cc polypropylene centrifuge tubes (Fischer Scientific, Pittsburgh, Pennsylvania) containing citrate-phosphate dextrose anticoagulant solution. It was then centrifuged at 4°C at 1,250 rpm for 20 min. The plasma was then separated from red cells and frozen at −70°C and stored at −20°C for 18 h to 24 h before further processing. The fresh frozen plasma was thawed in a 4°C cold room for 4 h and centrifuged at 2°C at 2,500 rpm for 40 min and plasma drained, leaving 1.0 to 1.5 ml of concentrated fibrinogen (20).

Sources of thrombin

Experimental animals

Thirty-six adult New Zealand white rabbits weighing 3.0 to 3.8 kg were used in these studies. Experimental atherosclerosis was induced by cholesterol diet and balloon injury as described previously (22). After two weeks of the atherogenic diet, the animals were anesthetized by a mixture of ketamine (10 mg/kg) and xylazine (1 mg/kg) administered intravenously and then maintained with pentobarbital (12 mg/kg intravenously) when required. The femoral artery was exposed and ligated, and the iliac arteries were deendothelialized by passing an inflated 4F balloon embolectomy catheter. The atherogenic diet was continued for four weeks before treatment.

On the day of the second procedure, the abdominal aorta from the iliac bifurcation to the femoral arteries was carefully exposed. Two treatment zones averaging 2.0 to 2.5 mm in diameter and 2 mm in length were identified by placing a fine prolene stitch in the adventitia. The distance from each stitch to the iliac bifurcation was measured and recorded.

The method of inducing atherosclerotic lesions is reproducible in our lab. Histological specimens obtained from control animals (n = 20) revealed significant lesions at 30 days after cholesterol diet and balloon injury. In another control group (with double balloon injury, n = 10) of animals harvested 60 days after second injury, two zones of neointima were present. In a majority of these animals internal elastic lamina was also injured. It should be noted that studies with Evans Blue revealed incomplete reendothelialization (46.5 ± 6.7%) in this group at 60 days. Blood cholesterol levels in these animals were elevated to 590 ± 72 mg/dl when compared with animals on a normal diet (190 ± 16 mg/dl).

Animals used in this study received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication #85-23, revised 1985).

Angiography and angioplasty

Angiography was performed through a 4 to 5F introducer sheath before and after balloon angioplasty of the iliac arteries. Immediately before introducing the catheters, a heparin bolus (1 mg/kg) was administered.

Balloon dilation was performed with a 3.0 mm balloon (length—20 mm). The inflation protocol consisted of two cycles of inflation to 8.0 atm for 30 s each, followed by one cycle of inflation to 8.0 atm for 90 s.

EC delivery

After angioplasty and angiography, one or two iliac arteries per rabbit were selected for EC seeding (group 1), EC/glue reconstruction (group 2) or glue only application (group 3). Next, the 2 ml of solution, containing ECs with or without glue or only glue were injected into the artery at 2 atm for 60 s at the rate of 0.5ml/min with custom made Wolinsky infusion balloon (3.0 mm diameter, 20 mm length) with eight holes. The size of each hole was 100 μm. The pressure of 2 atm very gently supports the delivery balloon against the target and permits the viscous liquid to be delivered to the arterial wall. The actual volume of solution delivered to the arterial surface under such conditions was 0.5 ml. Negative pressure was then applied to the balloon, and it was gently removed; a new balloon was then used to perform intervention into the contralateral artery according to the same protocol.

Concentration of the thrombin in the glue was 0.05 NIHU/ml, and the fibrinogen concentration was 2.5 mg/ml. This concentration of thrombin causes the fibrinogen to polymerize in 40 to 90 s. Such quick polymerization induced gelation of the delivery liquid right at the site of intervention and prevented possible migration of the delivered material downstream. The total number of ECs present in the matrix was 6 × 105 cells. In six animals the glue/EC matrix was labeled with the green or red fluorescent dye PKH26.

The EC/glue matrix or glue only were injected with a Microjet TM dual-syringe system. One syringe was filled with thrombin solution, and another contained fibrinogen with the mixture of ECs.

Acute studies: EC/glue reconstruction

After balloon angioplasty one of the iliac arteries was subjected to EC/glue reconstruction using a Wolinsky infusion balloon. The contralateral iliac artery was subjected to EC seeding alone with the same balloon type, but with the respective number of ECs suspended in DMEM.

The site of catheter introduction was repaired with one stitch using a prolene suture. After treatment, blood was allowed to perfuse the seeded arteries for 4 h to expose the newly attached ECs to physiologic shear forces. In this acute experiment, allogenic ECs were used. All 12 animals were sacrificed and subjected to macroscopic (n = 6) and histologic analysis (n = 6). In six animals the areas of endothelial denudation were assessed by staining with Evans blue dye (administered intravenously 30 min before sacrifice). The arteries were opened longitudinally and photographed en face for the measurement of the area with repaved endothelium, expressed as a percentage of the total area between bifurcation and femoral artery.

Chronic studies: impact on restenosis

Twelve rabbits were treated in the same fashion and restudied at 60 days after angioplasty/cell seeding. Treatment sites were assigned at random to either EC seeding or EC/glue groups. In these experiments autologous EC were used to avoid immunological response. An additional 10 rabbits were subjected to endoluminal reconstruction with glue only. At 60 days angiography was performed via aortic cannulation. The angiographic luminal diameter of the treatment zones was determined before sacrifice using a Stereoscope portable X-ray system with C-arm and monitoring system (CGR/General Electric, Milwaukee, Wisconsin). The data were recorded on videotape, and a permanent photographic image was obtained for each animal. The femoral arteries were then clamped, and all side branches of the iliac artery ligated. After fixation at hydrostatic pressure of 110 mm Hg for 60 min with 10% buffered formalin, the arteries were dissected to reveal the treatment zone.

The vessel was sectioned in 2 mm increments to produce a minimum of nine cross-sections. After embedding in paraffin and dehydration, the sections were stained for light microscopic analysis with: 1) hematoxylin and eosin, and 2) Movatt’s elastic tissue pentachrome.

Endothelial structure was studied using en face scanning electron microscopy. Segments of arteries were fixed in 3% glutaraldehyde for 24 h and then transferred to sodium cacodylate buffer. Dehydrated specimens were then dried in liquid CO2, sputter coated with gold and observed with a scanning electron microscope (Philips 500, Eindhoven, the Netherlands).

Analysis of data

Two experienced observers blinded to the origin of the samples and using a Tamaya Digital Planimeter (Lietz, Overland Park, Kansas) determined the cross-sectional areas of lumen, intimal hyperplasia and media. The borders of the external elastic lamina, internal elastic lamina, neointima and vessel lumen were traced from the photographs, and respective areas were calculated. Animals were excluded if an occlusive thrombus was detected. Interobserver variability was <8%. Vessel segments with maximal stenosis were selected for analysis. The extent of injury was assessed as described by Schwartz et al. (23).

The angiographic data were analyzed for changes to luminal diameter for all treatment zones immediately before and after treatment and 60 days before the harvest of the vessels. Two blinded observers recorded the diameter of each treatment zone by tracing the original image of the video recordings onto clear transparent sheets. This method is reproducible within 0.2 mm in our laboratory. For all treatment sites minimal luminal diameter (MLD) was determined. From these data acute gain and loss were derived.

Statistical evaluation

Data (mean ± standard deviation) were analyzed for overall differences between treatment groups by analysis of variance test for multiple comparisons followed by Bonferroni correction for multiple comparisons.

Results

Angioplasty and local delivery procedures were successfully accomplished in 30 rabbits. Two rabbits from the acute study group had major arterial dissection with extravasation of dye after angioplasty. Four rabbits from the chronic group had an occlusive thrombus at follow-up. One had thrombus formed in the iliac subjected to endothelial seeding procedure; the second rabbit had thrombus in the vessel that underwent glue/cell reconstruction, and the two animals with thrombotic occlusion had glue only delivered. All these animals were excluded from the final analysis.

EC/glue reconstruction—acute study

Macroscopic examination using Evan’s blue demonstrated circumferential attachment of the glue/EC matrix to 68.21 ± 6.77% of the denuded arterial wall in comparison with 13.85 ± 3.92% attachment after seeding with ECs alone (Table 1). These results were confirmed by the histologic study of fluorescence labeled ECs (Figs. 1 and 2). ⇓⇓ Attachment efficiency calculated from intensity of fluorescent stain PKM-26 extracted from seeded cells was very low in the arteries subjected to EC seeding alone (10.1 ± 8.8%). In contrast, the percentage of attached cells was much higher in arteries subjected to glue/cell reconstruction (75.3 ± 13.4%) (p < 0.0003). All vessels undergoing reconstruction remained patent and appeared grossly free of thrombus. Scanning electron microscopy of these arteries showed new ECs spreading within the fibrin strands (Fig. 3).

Percentage of cells attached to the vessel wall after endothelial cell/glue application and exposition to blood flow. Efficiency of attachment was calculated from intensity of fluorescent stain PKm-26 extracted from the transplanted cells.

Late loss in the EC seeding alone group was 1.12 ± 0.31 mm and 1.17 ± 0.29 mm in the glue only group compared with 0.69 ± 0.21 mm in the EC/glue group (p < 0.002 between EC and glue/cell and p < 0.003 between glue/cell and glue only interventions).

Histological evaluation

Plainimeter analysis showed that the luminal area was significantly greater in the EC/glue group (1.23 ± 0.35 mm2) than it was in the EC seeding group (0.65 ± 0.09 mm2) and glue only group (0.72 ± 0.41 mm2; Table 3). Similar significant differences were demonstrated in intimal area (0.76 ± 1.8 mm2—EC/glue group, 1.25 ± 0.26 mm2 EC group and 1.01 ± 0.53 mm2—glue only group; (p < 0.001 between EC seeding and glue/cell and p < 0.05 between glue/cell and glue only treatment groups; Table 3).

Discussion

Interventions on endothelium

Enhancement of endothelial regeneration has been addressed with a number of techniques, including EC seeding (24) and local delivery of growth factors (25–28). Although transplantation of ECs during or after coronary intervention is an attractive concept, three major limitations include: 1) prolonged seeding time, 2) optimal delivery device, and 3) adhesion of a functional EC to the area of vascular injury. In studies on swine femoral arteries, Nabel et al. (4) achieved 2 to 11% adherence of cells to the denuded arterial wall after 30 min of reseeding. Thompson et al. (5) have achieved 36% EC attachment to damaged human saphenous veins in vitro. The same group demonstrated 17% cell retention after 100 min of blood flow in previously angioplastied external iliac arteries of rabbits (6). Conte et al. (7) have shown that autologous venous cells can be genetically modified and returned to the surface of a balloon-injured rabbit femoral artery. In their experiments, vessels examined at four to seven days after seeding displayed 40% to 90% coverage with transduced cells, even when seeded at subconfluent density, and an intact EC monolayer, as evidenced by scanning electron microscopy studies. However, their method required surgical exposure of the vessels and complete interruption of blood flow for 30 min. This study demonstrated a method for quickly reattaching the glue/EC matrix to 68.0 ± 6.7% of the denuded arterial wall determined with Evans Blue technique. However, decreased Evans Blue staining may also reflect an independent effect of glue rather than integrity of transplanted endothelial barrier necessary to exclude Evans Blue staining. Therefore, we conducted additional experiments with fluorescence labeling of transplanted ECs. Attachment efficiency of ECs calculated from intensity of PKM-26 was significantly higher in arteries subjected to glue/cell reconstruction (Fig. 2). Scanning electron microscopy (Fig. 3) also confirmed these results.

In our experiments it was also found that all vessels undergoing reconstruction remained patent and appeared grossly free of thrombosis. Other studies have shown that EC seeding also reduces platelet deposition in a canine endarterectomy model at 5 h; 1, 2, 3 and 4 days and 4 weeks (24). Our study also shows that arterial reconstruction with EC/glue has significantly reduced restenosis in comparison with EC seeding or glue at follow-up (Figs. 4 and 5, Table 3). ⇓⇓

Movat’s stain. (A) Iliac artery of cholesterol-fed rabbit 60 days after balloon injury and local saline (control) application. Cross-section shows representative fibrocellular hyperplasia with focal macrophage/foam cell accumulation in the core. Waviness of both remaining internal and external laminae is suggestive of a remodeling process leading to shrinkage. (B) Contralateral iliac artery of the same animal after balloon injury and local application of glue. Cross-section shows comparable initial mechanical injury with fibrocellular hyperplasia without significant foam cell accumulation. Little inflammation and no angiogenesis are seen, but the tunica adventitia displays some reinforcing collagenous thickening in the upper left quadrant. (C) and (D) Iliac arteries from another cholesterol fed animal 60 days after procedure. On both iliac there is neointima around the entire circumference, but the internal elastic lamina is intact, and the original tunica media shows intimal cell loss and little compression, indicating that in these positions the initial injuries were present, symmetric and comparable. (C) The segment treated with endothelial cell seeding shows a concentric lesion with the fibrocellular intimal hyperplasia. (D) Local treatment with glue/cell reconstruction results in a minimal fibrocellular hyperplasia with little variation in thickness mostly due to the previous neointima. (A) to (D) Movat stain all taken with a digital camera at the same magnification and further processed with photofinish 4. L = lumen; M = media; N = neointima.

(A) Example of a severe lesion from another animal showing a significant breaking of the internal elastic lamina, the media and external elastic lamina, with very extensive neointimal hyperplasia and formation of a lipid rich core 60 days after treatment with endothelial cell seeding procedure. (B) Contralateral artery from the same animal with comparable level of injury. The intima in this glue/cell treated segment is moderate and hypercellular in nature.

Another approach to accelerate endothelialization is the local delivery of growth factors. Callow et al. (25) used vascular permeability factor, which is a naturally occurring growth factor, to stimulate EC proliferation in a rabbit model. Recently, Van Belle et al. (26) demonstrated that local delivery of vascular endothelial growth factor accelerated stent endothelialization and reduced stent thrombosis in a rabbit model. However, while growth factors increase the rate of endothelial repaving, many of them are also potent mitogens for vascular smooth muscle cells (25,26). Indeed, Moulton et al. (29) observed that prolonged treatment with angiogenesis inhibitors Endostatin or TNP-470 reduced plague growth in apo −/− mice. However, several experimental and human studies (26–30) with direct application of vascular endothelial growth factor protein or naked DNA to the injured arteries demonstrated no evidence of accelerated atherosclerosis.

Fibrin glue as a vehicle for endothelial delivery

It has been shown that ECs are able to grow faster on surfaces pretreated with plasma proteins, especially those involved in coagulation (31–33). Fibronectin, fibrinogen and vitronectin contained in our autologous cryoprecipitated preparations have specific binding sites for ECs (3). Our preliminary studies have confirmed that ECs remain attached to fibrin meshwork and are viable in the three-dimensional glue matrix (unpublished data). The noncytotoxic fibrin glue meshwork, in addition, is flexible and compliant, may be easily adapted to a circumferential contour wall and is timely resorbed to leave a completely healed tissue. The structure is characterized by areas occupied by adherent ECs and a cell-free area that may serve as a depot for drug delivery. In the cryoprecipitated plasma, the three-dimensional fibrin glue structure undergoes covalent cross-linking by the formation of glutamyl-lysil isopeptide bonds, which develop between the protein chains (3,13,17,18,31,33). Our results show very significant circumferential attachment of the glue/EC matrix to the denuded arterial wall when compared with seeding with ECs alone. In this experiment, the addition of fibrin glue to the ECs also decreased myointimal thickness, and the luminal area was significantly greater in the EC/glue group than it was in the EC-seeding group. In addition, the ECs from the border of the denuded area then preferentially migrate into this three-dimensional structure (32).

Furthermore, as an end point of thrombosis, a new fibrin layer may help limit new thrombosis and fibrin deposition. Indeed, we have previously demonstrated (33) that fibrin coating of stents resulted in reduced platelet adhesion in vitro and prevented stent thrombosis in an animal model. However, results of our present studies failed to demonstrate favorable influence of fibrin reconstruction without EC upon restenosis.

In the clinical setting this process would require the use of the patient as the autologous donor for the ECs and reagents for the biological glue. Thus, the method has the advantage of avoiding potential immunological/rejection problems. These data indicate that related plasma proteins are able to perform some of the functions of the extracellular matrix involved in anchoring ECs to the vessel wall.

Local delivery

One limitation of this technique is that the presence of side branches within the site of intervention limits efficacy of the seedling and may cause their loss. Secondly, the Wolinsky infusion balloon used in our studies is not optimal for glue/cell delivery (34). The limitation of this catheter is pressure driven local delivery, which causes additional vessel damage and relatively low efficacy of local delivery. The other catheter designs (35) may increase the efficacy of EC transplantation. One concept is a dual delivery catheter, one for thrombin solution and another for fibrinogen loaded with ECs. This design will also allow glue to polymerize directly on the surface of the arterial segment and will eliminate possible occlusion of the delivery holes of the catheter that make delivery more homogenous and minimize distal thrombosis.

Study limitations

One of limitations of this study is the animal model. The principal disadvantages of the atherosclerotic rabbit model include morphologic differences from human lesions.

Another limitation of the study was that the function of transplanted ECs is unknown, and additional studies are needed to address this issue.

Finally, it is difficult to differentiate the effect of EC/glue transplantation upon the integrity of the new endothelial layer from the independent effect of fibrin glue. However, the concept of reconstruction of the arterial wall with EC/glue matrix to prevent restenosis remains appealing.

Conclusions

We have demonstrated that: 1) vascular EC reconstruction using autologous EC/glue matrix is substantially more efficient than EC seeding alone and does not result in occlusive thrombus formation, 2) the degree of restenosis for angioplasty is favorably influenced by treatment with EC/glue matrix in an atherosclerotic rabbit. However, clinical trials are needed to further establish the efficacy of this intervention.

Acknowledgements

The authors are grateful to John Petersen, MD, for his review of the manuscript. The authors also wish to thank Cathy Kennedy for her editorial assistance on this manuscript.

Footnotes

☆ Supported, in part, by a research grant from Global Therapeutics, Inc., Broomfield, Colorado.

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